1. Technical Field
The present invention relates to a method for identifying genes that are translationally regulated. More specifically, the present invention relates to the rapid isolation of differentially expressed or developmentally regulated gene sequences through segregation of mRNAs into translated and untranslated pools and comparing the relative abundance of the mRNAs found in these pools by differential analysis.
2. Background Art
The identification and/or isolation of genes whose expression differs between two cell or tissue types, or between cells or tissues exposed to stress conditions, chemical compounds or pathogens, is critical to the understanding of mechanisms which underlie various physiological conditions, disorders, or diseases. Regulation of gene expression has been shown to play an important part in many biological processes including embryogenesis, aging, tissue repair, and neoplastic transformation. Gene regulation at the level of translation has been shown to be of critical importance. For example, it has been demonstrated that a group of mRNAs are stored in an egg as a pool of untranslated mRNAs which, following fertilization, shift into the pool of translated mRNAs. Another example of a change in the translational state of mRNA is a subgroup of mRNAs which code for heat shock proteins which are not translated under normal physiological conditions. These mRNAs begin to be translated following exposure of cells to high temperatures.
A number of methods have been developed for the detection and isolation of genes which are activated or repressed in response to developmental, physiological, pharmacological, or other cued events. One particular method is described in U.S. Pat. No. 5,525,471 to Zeng, is subtractive hybridization. Subtractive hybridization is a particularly useful method for selectively cloning sequences present in one DNA or RNA population but absent in another. The selective cloning is accomplished by generating single stranded complementary DNA libraries from both control cells/tissue (driver cDNA) and cell/tissue during or after a specific change or response being studied (tester cDNA). The two cDNA libraries are denatured and hybridized to each other resulting in duplex formation between the driver and tester cDNA strands. In this method, common sequences are removed and the remaining non-hybridized single-stranded DNA is enriched for sequences present in the experimental cell/tissue which is related to the particular change or event being studied. (Davis et al., 1987).
Currently used methodologies to identify mRNAs encoding proteins which are being induced/reduced following a cue or stimulus rely on changes in the mRNA levels following transcriptional induction/repression via screening of differentially expressed mRNAs. One such method for the identification of differentially expressed mRNAs is disclosed in U.S. Pat. No. 5,459,037 to Sutcliffe et al. According to this method, an mRNA population is isolated, double-stranded cDNAs are prepared from the mRNA population using a mixture of twelve anchor primers, the cDNAs are cleaved with two restriction endonucleases, and then inserted into a vector in such an orientation that they are anti-sense with respect to a T3 promotor within the vector. E. coli are transformed with the cDNA containing vectors, linearized fragments are generated from the cloned inserts by digestion with at least one restriction endonuclease that is different from the first and second restriction endonucleouseases and a cDNA preparation of the anti-sense cDNA transcripts is generated by incubating the linearized fragments with a T3 RNA polymerase. The cDNA population is divided into subpools and the first strand cDNA from each subpool is transcribed using a thermostable reverse transcriptase and one of sixteen primers. The transcription product of each of the sixteen reaction pools is used as a template for a polymerase chain reaction (PCR) with a 3'-primer and a 5'-primer and the polymerase chain reaction amplified fragments are resolved by electrophoresis to display bands representing the 3'-ends of the mRNAs present in the sample. This method is useful for the identification of differentially expressed mRNAs and the measurement of their relative concentrations. This type of methodology, however, is unable to identify mRNAs whose levels remain constant but their translatability is variable or changes.
Schena et al. developed a high capacity system to monitor the expression of many genes in parallel utilizing microarrays. The microarrays are prepared by high speed robotic printing of cDNAs on glass providing quantitative expression measurements of the corresponding genes (Schena et al., 1995). Differential expression measurements of genes are made by means of simultaneous, two color fluorescence hybridization. However, this method alone is insufficient for the identification of translationally regulated genes.
The use of a known inhibitor of hypusine formation, mimosime, was used to reversibly suppress the hypusine-forming deoxyhypusyl hydroxylase in cells while differentially displaying their polysomal versus non-polysomal mRNA populations. (Hanauske-Abel et al., 1995) Utilizing this method, several species of mRNA were discovered which disappear and reappear, respectively, at polysomes in connection with inhibition and disinhibition of hypusine formation and which are thought to code for translationally controlled enzymes. This method only teaches the use of a known stimulating element (i.e., inducer or repressor) to identify translationally regulated genes. This method does not provide a mechanism for the detection and/or identification of translationally regulated genes where the stimulating element is unknown.
Generally, the translation of eukaryotic mRNAs is dependent upon 5' cap-mediated ribosome binding. Prior to translation, the ribosome small sub-unit (40S) binds to the 5'-cap structure on a transcript and then proceeds to scan along the mRNA molecule to the translation initiation site where the large sub-unit (60S) forms the complete ribosome initiation site. In most instances, the translation initiation site is the first AUG codon. This "scanning model" of translation initiation accommodates most eukaryotic mRNAs. A few notable exceptions to the "scanning model" are provided by the Picornavirus family. These viruses produce non-capped transcripts with long (600-1200 nucleotides) 5'-untranslated regions (UTR) which contain multiple non-translation initiating AUG codons. Because of the absence of a cap structure, the translational efficiency of these RNAs is dependent upon the presence of specific sequences within the untranslated regions (UTR) known as internal ribosome entry sites (IRES).
More recently, IRES containing mRNA transcripts have been discovered in non-viral systems such as the mRNA encoding for immunoglobulin heavy chain binding protein, the antenapedia gene in Drosophila, and the mouse Fg1-2 gene. These discoveries have promoted speculation for the role of cap-independent translation in the developmental regulation of gene expression during both normal and abnormal processes.
The discovery of the above-mentioned non-viral IRES containing mRNAs implies that eukaryotic IRES sequences could be more wide spread than has been previously realized. The difficulty in identifying eukaryotic IRES sequences resides in the fact that they typically cannot be identified by sequence homology. [Oh et al., 1993; Mountford et al., 1995; Macejak et al., 1991; Pelletier et al., 1988; Vagner et al. 1995] It would, therefore, be advantageous to have a method for identifying IRES containing mRNA in order to identify translationally controlled genes operating via 5'-cap independent translation in order to ascertain and assess their association with both normal and abnormal processes.
Therefore, it would be desirable to have a rapid, reliable, and reproducible method for the identification and cloning of clinically and therapeutically relevant differentially expressed genes which will overcome the inherent problems associated with the prior art methods.